Guided bone regeneration membrane and manufacturing method thereof

ABSTRACT

Disclosed is a guided bone regeneration membrane including a novel mechanism that effectively induces a bone reconstruction ability. The mechanism is provided by forming a bi-layered structure of a first nonwoven fabric layer containing a silicon-releasable calcium carbonate and a poly(lactic acid) as principal components and a second nonwoven fabric layer containing a poly(lactic acid) as a principal component; and coating the first nonwoven fabric layer with an apatite. The guided bone regeneration membrane is available by using a nonwoven fabric manufacturing technique through electrospinning and a simulated body fluid soaking technique.

FIELD OF THE INVENTION

The present invention relates to a guided bone regeneration membrane anda manufacturing method thereof. The guided bone regeneration membrane isused in a guided bone regeneration (GBR) technique which is one oftechniques for repairing bone defects and which is widely used in thefield of oral surgery and maxillofacial surgery.

RELATED ART OF THE INVENTION

Guided bone regeneration membranes are masking membranes that cover bonedefect areas so as to prevent invasion of non-osteogenesis-contributedcells and tissues into the bone defect areas and to allow the bone toreconstruct by taking full advantage of self-regenerative power thereof.Guided bone regeneration techniques using these membranes cure bonedefects by using a healing potential which the living body inherentlyhas. The techniques are not complicated in their operative proceduresand have given many satisfactory outcomes in oral surgery.

The guided bone regeneration membranes are broadly grouped undernon-bioresorbable membranes and bioresorbable membranes. Apolytetrafluoroethylene (expanded polytetrafluoroethylene; ePTEF) hasbeen practically used as a material for a non-bioresorbable membrane,from which good clinical data have been obtained. This material,however, places a not-so light burden on a patient, because it is notbioresorbable and thereby needs a secondary operation for the removal ofthe membrane after the target bone defect area is repaired. In addition,it is difficult to adopt this material to a large defect area, becausethe material is bioinert (non-bioresorbable). In contrast, use of guidedbone regeneration membranes that are bioresorbable can avoid thesurgical stress caused by the secondary operation. Exemplary materialsfor such bioresorbable guided bone regeneration membranes includepoly(lactic acid)s as bioresorbable synthetic polyesters; andcopoly(lactic acid/glycolic acid)s; and collagens and fasciae each ofbiological origin. Such bioresorbable guided bone regeneration membraneshave been recently investigated and developed heavily, and some of themhave already been commercialized. Typically, there have been proposed awide variety of guided bone regeneration membranes and manufacturingmethods thereof; such as a bone regeneration membrane including acomposite of a bioresorbable polymer with tricalcium phosphate orhydroxyapatite and having micropores (Japanese Unexamined PatentApplication Publication (JP-A) No. H06 (1994)-319794); a protectivemembrane including a felt made from a bioresorbable material (JapaneseUnexamined Patent Application Publication (JP-A) No. H07 (1995)-265337;and Japanese Unexamined Patent Application Publication (JP-A) No.2004-105754); a multilayer membrane including a sponge-like collagenmatrix layer and a relatively impermeable barrier layer (JapaneseUnexamined Patent Application Publication (Translation of PCTApplication) (JP-A) No. 2001-519210); a bioresorbable tissueregeneration membrane for dental use, which has a porous sheet-likestructure including a polymer blend containing two or more differentbioresorbable polymers (Japanese Unexamined Patent ApplicationPublication (JP-A) No. 2002-85547); a resorbable flexible implant in theform of a continuous micro-porous sheet (Japanese Unexamined PatentApplication Publication (Translation of PCT Application) (JP-A) No.2003-517326); and a biocompatible membrane prepared by three-dimensionalpowder sinter molding through application of laser light to abiodegradable resin powder (Japanese Unexamined Patent ApplicationPublication (JP-A) No. 2006-187303).

In particular, oral or maxillary bone defects should be desirably curedas soon as possible, because it is very important to maintain and ensuremastication for the health maintenance in a super-graying society. Toimprove osteogenic ability, there have been attempts to incorporate to abioresorbable membrane a factor such as an osteogenesis inducer(Japanese Unexamined Patent Application Publication (JP-A) No. H06(1994)-319794), a growth factor or a bone morphogenic protein (JapaneseUnexamined Patent Application Publication (Translation of PCTApplication) (JP-A) No. 2001-519210; and Japanese Unexamined PatentApplication Publication (JP-A) No. 2006-187303). However, it isdifficult to handle these factors. Accordingly, demands have been madeto develop a bioresorbable guided bone regeneration membrane havingsuperior bone reconstruction ability to allow the bone toself-regenerate more reliably and more rapidly.

In view of recent trends of researches and technologies for bio-relatedmaterials, the main stream of researches has been shifted from amaterials design for the bonding of a material with the bone to amaterials design for the regeneration of a real bone; in theseresearches, the role of silicon in osteogenesis has been receivedattention; and there have been designed a variety of materialscontaining silicon (TSURU Kanji, OGAWA Tetsuro, and OGUSHI Hajime,“Recent Trends of Bioceramics Research, Technology and Standardization”,Ceramics Japan, 41, 549-553 (2006)). For example, there has beenreported that the controlled release of silicon genetically acts oncells to promote osteogenesis (H. Maeda, T. Kasuga, and L. L. Hench,“Preparation of Poly(L-lactic acid)-Polysiloxane-Calcium CarbonateHybrid Membranes for Guided Bone Regeneration”, Biomaterials, 27,1216-1222 (2006)). Independently, when composites of a poly(lactic acid)with one of three calcium carbonates (calcite, aragonite, and vaterite)are soaked in a simulated body fluid (SBF), the composite of apoly(lactic acid) with vaterite forms a bone-like apatite within ashortest time among the three composites (H. Maeda, T. Kasuga, M.Nogami, and Y Ota, “Preparation of Calcium Carbonate Composite and TheirApatite-Forming Ability in Simulated Body Fluid”, J. Ceram. Soc. Japan,112, S804-808 (2004)). These findings demonstrate that the use ofvaterite which gradually releases silicon is believed to be a key toprovide a guided bone regeneration membrane that gives rapid bonereconstruction.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a bioresorbable guidedbone regeneration membrane that includes a novel mechanism effectivelyinducing a bone reconstruction ability. Another object of the presentinvention is to provide a method for manufacturing a guided boneregeneration membrane of high performance (achieving rapid bonereconstruction) in an inexpensive and industrially advantageous manner.

The present invention provides, in an embodiment, a guided boneregeneration membrane which has a bi-layered structure including a firstnonwoven fabric layer and a second nonwoven fabric layer. The firstnonwoven fabric layer contains a silicon-releasable calcium carbonate(Si—CaCO₃) and a biodegradable resin, represented by a poly(lactic acid)(PLA), as principal components (hereinafter referred to as “Si—CaCO₃/PLAlayer”). The second nonwoven fabric layer contains biodegradable resin,representedbya PLA, as a principal component (hereinafter referred to as“PLA layer”). In the guided bone regeneration membrane, the Si—CaCO₃/PLAlayer may be further coated with an apatite.

The PLA layer has the function of preventing the invasion of softtissues, and the apatite-coated Si—CaCO₃/PLA layer has the function ofimproving cellular affinity and/or osteogenic ability. In anotherembodiment, a technique of manufacturing a nonwoven fabric throughelectrospinning is adopted to the manufacturing of such a guided boneregeneration membrane. This provides an easy manufacturing of a membranethat has continuous pores for supplying nutrients to cells and showsimproved fitting ability to an affected area. Such a bioresorbableapatite that improves cellular initial adhesion can be easily applied tothe Si—CaCO₃/PLA layer by soaking the layer in a simulated body fluid(SEF).

The guided bone regeneration membrane according to the present inventionshows high cellular growth ability in cellular affinity tests usingosteoblastic cells (MC3T3-E1 cells) and is expected as a bioresorbableguided bone regeneration membrane that excels in bone reconstructionability. The method according to the present invention can easily andefficiently manufacture a guided bone regeneration membrane having theabove possibility.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, features and advantages of the present invention will beunderstood more fully from the following detailed description made withreference to the accompanying drawings. In the drawings:

FIG. 1 is a scanning electron micrograph (SEM photograph) of a PLA layersurface of a guided bone regeneration membrane prepared in Example 1;

FIG. 2 is a scanning electron micrograph of a Si—CaCO₃/PLA layer surfaceof the guided bone regeneration membrane prepared in Example 1;

FIG. 3 is a scanning electron micrograph of a surface of a PLA layerprepared in Example 2;

FIG. 4 is a scanning electron micrograph of a surface of a Si—CaCO₃/PLAlayer prepared in Example 2;

FIG. 5 is a scanning electron micrograph of fibers configuring theSi—CaCO₃/PLA layer prepared in Example 2;

FIG. 6 is a scanning electron micrograph of fibers configuring theSi—CaCO₃/PLA layer after soaking a composite membrane prepared inExample 2 in 1.5 SBF;

FIG. 7 depicts X-ray diffraction patterns of the composite membraneprepared in Example 2, before and after soaking in 1.5SBF; and

FIG. 8 is a graph for the evaluation of the cellular affinity of theSi—CaCO₃/PLA layer and PLA layer prepared in Example 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be described further with reference tovarious embodiments in the drawings.

First Embodiment

According to a preferred embodiment of the present invention, such aguided bone regeneration membrane can be manufactured through the stepsof electrospinning and soaking in a simulated body fluid (SBF). In theelectrospinning step, a positive high voltage is applied to a polymersolution, and the resulting polymer solution is sprayed as fibers to anegatively charged collector.

A spinning solution for the formation of the PLA layer (PLA spinningsolution) is prepared by dissolving a poly(lactic acid) in chloroform(CHCl₃) or dichloromethane (DCM). The PLA spinning solution preferablyhas a poly (lactic acid) concentration of from 4 to 12 percent by weightfor easy spinning. In this connection, the poly(lactic acid) generallyhas a molecular weight of from about 20×10⁴ to about 30×10⁴. Formaintaining conditions for satisfactory spinning, the PLA spinningsolution may further contain dimethylformamide (DMF) and/or methanol(CH₃OH) in an amount up to about 50 percent by weight relative to theamount of CHCl₃ or DCM. Another spinning solution for the formation ofthe Si—CaCO₃/PLA layer (Si—CaCO₃/PLA spinning solution) is prepared byadding Si—CaCO₃ to the PLA spinning solution. The Si—CaCO₃ is preferablyadded to the solution so that the Si—CaCO₃/PLA layer has a Si—CaCO₃content of from 40 to 60 percent by weight. This allows an apatite todeposit efficiently on Si—CaCO₃/PLA fibers in the SBF soaking step.Alternatively, a Si—CaCO₃/PLA spinning solution can be prepared bykneading a poly (lactic acid) and Si—CaCO₃ in predetermined proportionsusing a heating kneader to give a composite, and dissolving thecomposite in a solvent. The Si—CaCO₃ may be prepared, for example, bythe method described in Japanese Patent Application No. 2006-285429(corresponding to Japanese Unexamined Patent Application Publication(JP-A) No. 2008-100878). The PLA layer preferably contains a poly(lacticacid) (PLA) alone or a copolymer between a poly (lactic acid) and apoly(glycolic acid) (PGA) (copoly(lactic acid/glycolic acid)) Exemplaryother biodegradable resins usable herein include synthetic polymers suchas polyethylene glycols (PEGS), polycaprolactones (PCLs), as well ascopolymers among lactic acid, glycolic acid, ethylene glycol, and/orcaprolactone; and natural polymers such as fibrin, collagens, alginicacids, hyaluronic acids, chitins, and chitosans. Each of these can beused instead of the PLA component in the Si—CaCO₃/PLA layer. TheSi—CaCO₃/PLA layer and the PLA layer may further contain inorganicsubstances that are usable without biological problems. Examples of suchinorganic substances include tricalcium phosphate, calcium sulfate,sodium phosphate, sodium hydrogenphosphate, calcium hydrogenphosphate,octacalcium phosphate, tetracalcium phosphate, calcium pyrophosphate,and calcium chloride.

Using an electrospinning apparatus, each of the PLA layer spinningsolution and the Si—CaCO₃/PLA spinning solution is charged and sprayedfrom a nozzle, converted into fibers in an electric field whileevaporating the solvent, the charged fibers are jetted toward acollector on a negative electrode and form a thin layer of fibers on thecollector. A desired guided bone regeneration membrane can be preparedby changing spinning conditions such as the concentration, solvent type,and supply speed (feed rate) of the spinning solution; spinning time;applied voltage; and distance between the nozzle and the collector. Theprepared nonwoven fabrics may be pressed so as to be compacted or tohave a desired thickness. A guided bone regeneration membrane having abi-layered structure is configured by spraying the PLA spinning solutionto form a PLA layer, and thereafter spraying the Si—CaCO₃/PLA spinningsolution to form a Si—CaCO₃/PLA layer on the PLA layer; or by preparinga PLA nonwoven fabric and a Si—CaCO₃/PLA nonwoven fabric independently,and combining the two nonwoven fabrics. The guided bone regenerationmembrane having a bi-layered structure is cut to a desired size andsoaked in a simulated body fluid (SBF) or a solution with 1.5 timeshigher concentration of inorganic ions compared to SBF (1.5SBF) at about37° C. for a predetermined time to precipitate an apatite on theSi—CaCO₃/PLA layer. This gives a bioresorbable guided bone regenerationmembrane including a novel mechanism that effectively induces the bonereconstruction ability. The SBF soaking can be performed even after thecombining (or laminating) the two layers. Even in this case, the apatitedeposits substantially not on the PLA layer but selectively on theSi—CaCO₃/PLA layer. This is because silicon contained in theSi—CaCO₃/PLA layer induces nucleation of apatite, and the calciumcomponent dissolves out to abruptly increase the degree ofsupersaturation of apatite, and the apatite selectively deposits on thesurface of the Si—CaCO₃/PLA layer; but the surface of the PLA layer ishydrophobic to avoid the deposition of apatite substantially.

EXAMPLES

Manufacturing methods of guided bone regeneration membranes according toembodiments of the present invention will be illustrated with referenceto several examples below. It should be noted, however, that theseexamples are included merely to aid in the understanding of the presentinvention and are not to be construed to limit the scope of the presentinvention.

Raw materials used in the examples are as follows.

Silicon-releasable calcium carbonate (Si—CaCO₃): Vaterite having asilicon content of 2.9 percent by weight and prepared by using slakedlime (Microstar T; purity 96% or more; Yabashi Industries Co., Ltd.,Japan), methanol (analytical grade reagent; purity 99.8% or more;Kishida Chemical Co., Ltd., Japan), γ-aminopropyltriethoxysilane (TSL8331; purity 98% or more; GE Toshiba Silicones Co., Ltd., Japan), andcarbon dioxide gas (high-purity liquefied carbon dioxide gas; purity99.9%; Taiyo Kagaku Kogyo K.K.)

Poly(lactic acid) (PLA): PURASORB PL Poly(L-lactide), molecular weightof 20×10⁴ to 30×10⁴, PURAC Biochem

Chloroform (CHCl₃): Analytical grade reagent, purity 99.0% or more,Kishida Chemical Co., Ltd., Japan

N,N-Dimethylformamide (DMF): Analytical grade reagent, purity 99.5% ormore, Kishida Chemical Co., Ltd., Japan Example 1

A PLA spinning solution having a PLA concentration of 6.8 percent byweight was prepared by blending 10 g of PLA, 110 g of CHCl₃, and 27.5 gof DMF. Independently, a Si—CaCO₃/PLA spinning solution having aSi—CaCO₃ concentration of 7.5 percent by weight and a PLA concentrationof 5.0 percent by weight was prepared by blending 15 g of Si—CaCO₃, 10 gof PLA, 140 g of CHCl₃, and 35 g of DMF. Using the prepared spinningsolutions, a guided bone regeneration membrane having a bi-layeredstructure of nonwoven fabrics was manufactured through electrospinning.

[PLA Layer Preparation Conditions]

Spinning solution feed rate: about 0.1 ml/min., applied voltage: 15 kV,distance between the nozzle and collector: 10 cm, nozzle: laterallymoves in a width of 3 to 4 cm at a rate of 15 cm/min, conveyor-typecollector (conveyor speed: 5 to 6 m/min), spinning time: about 170minutes

[Si—CaCO₃/PLA Layer Preparation Conditions]

Spinning solution feed rate: about 0.16 ml/min, applied voltage: 20 kV,distance between the nozzle and collector: 10 cm, nozzle: laterallymoves in a width of 3 to 4 cm at a rate of 15 cm/min, conveyor-typecollector (conveyor speed: 5 to 6 m/min), spinning time: about 130minutes

The microstructure of the prepared PLA layer (side for preventing softtissue invasion) is shown in the scanning electron microscope (SEM)photograph of FIG. 1. The microstructure of the Si—CaCO₃/PLA layer (boneregeneration side) is shown in the scanning electronmicrograph of FIG.2, demonstrating that Si—CaCO₃ particles are attached to PLA fibers.

Example 2

A spinning solution having a PLA concentration of 9.0 percent by weightwas prepared by blending 9 g of PLA and 91 g of CHCl₃, and using thisspinning solution, a PLA layer was prepared through electrospinning.

[PLA Layer Preparation Conditions]

Spinning solution feed rate: 0.05 ml/min, applied voltage: 20 kV,distance between the nozzle and collector: 15 cm, nozzle: fixed, platecollector: fixed, spinning time: 60 minutes

Independently, PLA and Si—CaCO₃ were kneaded in a heating kneader at200° C. for 15 minutes to give a Si—CaCO₃/PLA composite containing 60percent by weight of Si—CaCO₃. A spinning solution having a Si—CaCO₃concentration of 13.0 percent by weight and a PLA concentration of 8.7percent by weight was prepared by blending 25 g of the Si—CaCO₃/PLAcomposite and 90 g of CHCl₃, and using this spinning solution, aSi—CaCO₃/PLA layer was prepared through electrospinning.

[Si—CaCO₃/PLA Layer Preparation Conditions]

Spinning solution feed rate: 0.05 ml/min, applied voltage: is 20 kV,distance between the nozzle and collector: 15 cm, nozzle: fixed, platecollector: fixed, spinning time: 30 minutes

The two nonwoven fabrics prepared by the above procedures were each cutto a desired size and affixed or combined with each other to give onemembrane. Specifically, the PLA layer was laid over the Si—CaCO₃/PLAlayer, and a stainless steel mesh (40-mesh) was laid over the PLA layer.A plate heated at 150° C. to 160° C. was placed on the stainless steelmesh and pressed under a suitable pressure for about 10 seconds to givethe combined membrane (composite membrane). The scanning electronmicrographs of the PLA layer surface and of the Si—CaCO₃/PLA layersurface are shown in FIG. 3 and FIG. 4, respectively. The scanningelectron micrograph of fibers configuring the Si—CaCO₃/PLA layer isshown in FIG. 5, demonstrating that Si—CaCO₃ particles are attached toPLA fibers.

The Si—CaCO₃/PLA layer surface of the resulting composite membrane wasbrought into contact with 1.5SBF at 37° C. for one day. The scanningelectron micrograph of fibers on the side in contact with 1.5SBF isshown in FIG. 6, demonstrating that a substance quite different fromSi—CaCO₃ covers the surface of fibers, as compared to FIG. 5. The X-raydiffraction patterns before and after soaking in 1.5SBF are shown inFIG. 7, indicating that peaks of apatite appear after the soaking. Theseresults demonstrate that the Si—CaCO₃/PLA layer surface is coated withapatite.

FIG. 8 shows how cellular numbers (in terms of cellular numbers per 1cm²) vary after the inoculation of osteoblastic cells on theapatite-coated Si—CaCO₃/PLA layer surface (Si-composite), on the PLAlayer surface (PLA), and on a control (Thermanox: plastic disc for cellculture which has been treated on its surface). The data in FIG. 8demonstrate that the layer including PLA in combination with a novelmechanism gives higher growth capability to osteoblasts, and theresulting guided bone regeneration membrane is expected as abioresorbable guided bone regeneration membrane that excels in bonereconstruction ability.

Experimental Conditions

Cultivation using 24-well plate

Cell type; murine osteoblastic cells (MC3T3-E1 cells: Riken Institute ofPhysical and Chemical Research)

Cellular inoculation number: 1×10⁴ cells/well

Medium: α-MEM (containing 10% fetal bovine serum)

Medium exchange: on the day following the inoculation, thereafter everyother day

Cell counting method: The measurement was performed using the CellCounting Kit-8 (cellular growth/cellular toxicity analytical reagent;Dojindo Laboratories) in accordance with the protocol attached to thereagent.

While the above description is of the preferred embodiments of thepresent invention, it should be appreciated that the invention may bemodified, altered, or varied without deviating from the scope and fairmeaning of the following claims.

1. A guided bone regeneration membrane comprising a bi-layered structureincluding a first nonwoven fabric layer and a second nonwoven fabriclayer, the first nonwoven fabric layer containing a silicon-releasablecalcium carbonate and a biodegradable resin as principal components, andthe second nonwoven fabric layer containing a biodegradable resin as aprincipal component.
 2. The guided bone regeneration membrane accordingto claim 1, wherein the surface of the first nonwoven fabric layercontaining a silicon-releasable calcium carbonate and a biodegradableresin as principal components is coated with an apatite, the apatitehaving been deposited through soaking in a simulated body fluid.
 3. Theguided bone regeneration membrane according to claim 2, wherein thebiodegradable resin is a poly(lactic acid).
 4. The guided boneregeneration membrane according to claim 2, wherein the biodegradableresin is a poly(lactic acid).
 5. The guided bone regeneration membraneaccording to claim 1, wherein the silicon-releasable calcium carbonateis of vaterite phase.
 6. The guided bone regeneration membrane accordingto claim 2, wherein the silicon-releasable calcium carbonate is ofvaterite phase.
 7. The guided bone regeneration membrane according toclaim 3, wherein the silicon-releasable calcium carbonate is of vateritephase.
 8. The guided bone regeneration membrane according to claim 4,wherein the silicon-releasable calcium carbonate is of vaterite phase.9. A method for manufacturing a guided bone regeneration membrane, themethod comprising the steps of: forming a first nonwoven fabric throughelectrospinning, the first nonwoven fabric containing asilicon-releasable calcium carbonate and a biodegradable resin asprincipal components; and forming a second nonwoven fabric throughelectrospinning, the second nonwoven fabric containing a biodegradableresin as a principal component.
 10. The method for manufacturing aguided bone regeneration membrane, according to claim 9, wherein thebiodegradable resin is a polylactic acid).
 11. The method formanufacturing a guided bone regeneration membrane, according to claim 9,wherein the silicon-releasable calcium carbonate is of a vaterite phase.12. The method for manufacturing a guided bone regeneration membrane,according to claim 10, wherein the silicon-releasable calcium carbonateis of a vaterite phase.